Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source
Fig 4
Proposed mechanisms of synthesis-dependent strand annealing (SDSA) and mismatch repair.
(A) Double-stranded break caused by Cas9 is followed by 5’–3’ resection of the broken ends, leaving 3’ overhangs which will search for homology to initiate repair. (B) One of the overhanging strands finds homology in the donor and invades very closely to the homology arm-transgene junction, thereby bypassing the recoding on this side of the homology arms. The invading strand synthesises DNA using donor sequences as a template. (C) When this newly synthesised strand recognises homology on the 3’ overhang of the other end of the DSB, it dissociates from the template, anneals to the 3’ overhang, and is ligated to the 5’ end of one of the previously resected strands (squared in blue). (D) The other strand now repairs its break by DNA synthesis and ligation using the invading strand as its template, forming a heteroduplex region (squared in red). (E) Finally, the heteroduplex region is resolved by the mismatch repair pathway which favours the invading strand over the non-invading strand.